Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase

Abstract

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

Figure 1.

(a) Prototypical NOCAP synthase biosynthetic gene cluster from N. puris. (b) Biosynthesis of 1 by the NOCAP synthase. Key: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; KR, ketoreductase; ER, enoylreductase; cMT, C-methyltransferase; ACP, acyl carrier protein; TR, thioester reductase; and TEII, thioesterase II.

Figure 2.

In vitro characterization of modules L, 1, and 2 and tAT-TEII. (a) Extracted ion chromatograms (EICs) of 11⁺ charge state for malonyl-S-ACP_L and acetyl-S-ACP_L in the presence or absence of: KS_L. (b) EICs of malonyl-PPant and sorbyl-PPant ejected from malonyl-S-ACP₂ and sorbyl-S-ACP₂, respectively, in the presence or absence of module L. (c) EICs of 11⁺ charge states for acetyl-S-ACP₁ and holo-ACP₁ in the presence or absence of either tAT or tAT-TEII

Figure 3.

Reconstitution of the NOCAP synthase (a) in vitro and (b) in E. coli. (c) EICs of 1 and 2 from either in vitro reactions or E. coli pellet extracts. As a negative control, modules 1 and 2 (as one bimodular protein) were omitted.

Figure 4.

Structures of 1 and 2 assembled from 2D NMR data.